Hepatitis B virus (HBV) is the most prevalent human tumor virus. It is estimated that chronic HBV infection causes 1,000,000 cases of liver cancer annually. HBV actively replicates within liver cells to maintain its chronic infection. We work to understand the mechanisms by which HBV replicates in order to identify targets for new therapies. During this last funding period our most significant findings and progress included: (1) learning that HBV genome replication requires complex contributions from the replication template. The genomic RNA and DMA replication intermediates are not passive templates, but proceed through dynamic topologies that guide their own replication; (2) elucidating the involvement of the host cellular multivesicular body pathway in HBV virion production; and (3) conducting the first high throughput genetic screen to systematically identify host genes involved in HBV replication. This present proposal will build upon progress engendered during the last funding period. This project has three aims: (1) to study the synthesis of plus-strand DNA to elucidate (a) the mechanisms of template switching and inhibition of in situ priming, (b) the role of the C-terminus of capsid protein, and (c) the relationship between genome maturation and capsid envelopment; (2) to study the synthesis, accumulation and maintenance of cccDNA; and (3) to study the role of the Rho GTPase pathway in HBV virion production. These studies will uncover new mechanisms of virus replication and identify targets for new and better HBV therapies.